1. Field of the Invention
The invention relates to methods of generating mature dendritic cells with enhanced IL-12 production and compositions and systems for such dendritic cells.
2. Discussion of the Background
Dendritic cells (DCs), the most potent antigen presenting cells, are effective inducers of protective immunity against infectious diseases and cancer (Banchereau & Steinman 1998). The adjuvant function(s) of DCs has prompted intense interest in the use of DCs as a vaccine component, particularly after the advent of in vitro methods to generate large numbers of DCs from monocytes (Peters et al 1993, Sallusto & Lanzavecchia 1994). Over the past years, DC-based vaccines have been increasingly applied in the clinical treatment of cancer patients (Steinman et al., 2001; Parmiani et al., 2002). Following the initial success of the multi-epitope melanoma trial (Nestle et al 1998; 30% objective clinical responses), DCs have been used successfully to treat patients with melanoma, lymphoma and renal cell carcinoma (reviewed: Steinman et al., 2001; Parmiani et al., 2002). However, the overall clinical response rates do not exceed the predictable 15% observed for alternate immunotherapies (idem), which is below expectations, highlighting the need for improved design of DC-based vaccines, including the selection of the most appropriate types of DCs.
Although some of the early studies with DC-based vaccines successfully used FCS-based protocols (Nestle et al., 1998), the need to obtain the vaccine-applied DCs in possibly best-defined conditions (and to overcome potential reproducibility and regulatory issues) prompted the development of serum-free approaches to grow DCs.
Extensive research of recent years convincingly demonstrated that the effective induction of anti-tumor CTL responses requires the participation of fully-mature DCs because immature DCs are either ineffective, poorly immunogenic, or induce undesirable IL-10-producing regulatory T cells (Jonuleit et al 2000, Dhodapkar et al 2001). These considerations, in conjunction with the desire to use the most strictly-defined and reproducible conditions of DC generation for human use, established the dominant position of the “complete cytokine cocktail” composed of the combination of inflammatory cytokines IL-1β, TNFα, IL-6, and PGE2 (Jonuleit et al. 1997), as the “gold standard” of DCs used in cancer immunotherapy.
Fully-mature DCs induced by the combination of inflammatory cytokines IL-1β, TNFα, IL-6, and PGE2 (Jonuleit et al. 1997) have been consistently observed as superior to immature DCs in promoting a higher degree of specific T cell priming in vitro and in vivo (Jonuleit et al., 2001, Schuler-Thumer et al., 2000, Schuler-Thumer et al., 2002, Thurner et al., 1999, Dhodapkar et al., 2001).
Unfortunately, the maturation stage of DCs obtained in the currently-available protocols inversely correlates with their ability to produce IL-12p70 (Kalinski et al., 1999, Langenkamp et al., 2000), the cytokine with powerful anticancer Th1- and CTL-inducing properties (Trinchieri, 1998b); (Shurin et al., 1997).
Induction of Ag-specific CD8 T cells and Th 1-type CD4 T cells depends on the ability of DCs to provide CD4 and CD8 T cell precursors with high levels of co-stimulation and with interleukin-12 (IL-12), the major DC-produced anti-tumor cytokine. Previous work with DC transduced with IL-12 genes demonstrated that high IL-12-producing DCs are effective inducers of tumor rejection in experimental animals. However, use of IL-12 transduced DC in humans creates substantial logistic problems. It also carries potential risks associated with the administration of genetically-manipulated material and the risks of direct IL-12 toxicity and of deregulating the immune system due to uncontrolled IL-12 production.
Many have attempted to generate DC's using a variety of methods. For example, U.S. Pat. Nos. 5,851,756, 5,994,126 and 5,475,483 (Steinman, Inaba and Schuler) disclose methods for generating DCs from proliferating precursors and their maturation. Further, U.S. Pat. No. 5,866,115 discloses a method of developing DCs from DC34+ blood progenitors and U.S. Pat. Nos. 6,228,640 and 6,251,665 disclose a means of loading DCs developed from CD34+ progenitors with RNA or its expression products as a mean of achieving the expression of tumor-related or other target-related antigens. Similarly, U.S. Pat. No. 6,121,044 teaches a means of developing DC in bulk monocytes-depleted PBMC cultures. These patents focus on particular methods of generating immature dendritic cells rather than the particular conditions of the maturation of dendritic cells. More importantly, none of these patents disclose or teach the generation of dendritic cells with the unique properties described in the present invention. Specifically, none of the patents disclose or teach the combination of type I and type II interferons (such as IFNα and IFNγ), as a part of the cytokine cocktail used to produce fully mature DCs with high IL-12 producing capacity.
Thus, despite the efforts of many, the desirable combination of high immunostimulatory activity with a high capacity to produce IL-12p70 could not be attained by all previous DC-based vaccines which have employed either mature DCs exhibiting high stimulatory/low IL-12-secreting functions or immature DCs that display low stimulatory/high IL-12 secretion functions.
It is known in the art that the presence of IFN-γ during the either LPS-induced or IL-1β/TNFα-induced DC maturation, results in the induction of stable type-1 polarized DCs (DC1s) that produce up to 100-fold higher levels of IL-12p70 in response to subsequent CD40L stimulation or the interaction with CD40L-expressing CD4+ Th cells (Vieira et al., 2000, Mailliard et al., 2002). Unfortunately, the original DC1-inducing cytokine cocktail, composed of IL-1β, TNFα, and IFN-γ (Vieira et al 2000), does not allow for the induction of DC1s in serum-free media, which is desirable for clinical application.
DCs in the periphery can be exposed to a variety of environmental “triggers” that result in DC “maturation” and upregulation of factors critical to antigen-specific T-cell activation, including IL-12 production. In some cases, these signals are transmitted through Toll-like receptor (“TLRs”) and other cell-surface receptors expressed by DCs.
It is an object of the present invention to provide a means of triggering DC maturation through innate signaling pathways to enable DCs to express potent DC1-type function, regardless of the presence of factors present in serum, enabling in vitro derivation of DC1s for clinical applications.
It is an object of one preferred embodiment of the present invention to add at least one from the group of IFNα or IFNβ (type I interferons) or a type I interferon inducing factor such as polyinosinic:polycytidylic acid (poly-I:C) to the “classical” DC1-inducing cocktail (INFα/IL-1β/IFNγ) and to provide a means for generating fully-mature DC1s in serum-free AIM-V medium.
It is further an object of the present invention to provide an αlpha-type-1 DC to induce up to 50-fold higher levels of cancer-specific CTLs, and higher cytolytic activity of Th1 or NK cells compared to the current “gold standard” DCs (matured by IL-1β/TNFα/IL-6/PGE2; Jonuleit et al., 1997).